Jump to

Abstract

Ischemic heart disease (IHD) is the number one cause of morbidity and mortality in the US. We hypothesize that short hairpin RNA interference therapy can be used for treatment of IHD and this process can be followed noninvasively by molecular imaging. HIF-1α is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Here we focused on prolyl hydroxylase-2 (PHD2) gene as a potential knock-down target for inducing angiogenesis. PHD2 was cloned from mouse embryonic stem cells by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted after H1 promoter. CMV promoter driving a double fusion reporter gene (Fluc-GFP) cassette was inserted into shRNA plasmid (Figure 1⇓). This construct was used to transfect mouse C2C12 myoblast cell line. Compared to the scramble sequence control, inhibition of PHD2 increased levels of HIF-1α and several downstream angiogenic genes and proteins as assessed by semi-quantitative RT-PCR and Western blots. Using the densitometric analysis software, several angiogenesis genes were upregulated by >30% (P<0.01). Afterwards, the shRNA targeting PHD2 plasmid was injected intramyocardially following LAD ligation in mice. Animals were randomized into shRNA group (n=20) versus control group with scramble sequence injection (n=20). Longitudinal bioluminescence imaging detected expression of Fluc activity for 6 – 8 weeks. Echocardiographic study showed the shRNA group had improved fractional shortening comapred to the scramble group at week 2 (30%±5.2 vs. 23%±3.1; P<0.05). Taken together, this is the first study to image the biological role of shRNA therapy for improving cardiac function.